JP2012232948A - Anti-morphine antibody, method for measuring morphine using the same, and morphine measuring kit containing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a superior anti-morphine antibody capable of recognizing morphine specifically, and having low recognition ability to heroin, codeine, cocaine and ketamine, and to thereby facilitate measurement of morphine in a sample.SOLUTION: This anti-morphine antibody to be prepared is selected from a group comprising a single-chain antibody (scFv), a Fab fragment, a F(ab')fragment, a Fv fragment and a perfect antibody, which comprise anti-morphine antibody molecules capable of recognizing morphine specifically, having low recognition ability to heroin, codeine, cocaine and ketamine, and containing a heavy chain variable region and a light chain variable region, and further the anti-morphine antibody has, in the heavy chain variable region, (a) an amino acid sequence described on the 31th place to the 35th place of SEQ ID NO.1 in the sequence table as CDR-H1, (b) an amino acid sequence described on the 50th place to the 65th place of SEQ ID NO.1 as CDR-H2, and (c) an amino acid sequence described on the 98th place to the 110th place of SEQ ID NO.1 as CDR-H3, and/or also has, in the light chain variable region, (a) an amino acid sequence described on the 160th place to the 174th place of SEQ ID NO.1 in the sequence table as CDR-L1, (b) an amino acid sequence described on the 190th place to the 196th place of SEQ ID NO.1 as CDR-L2, and (c) an amino acid sequence described on the 229th place to the 237th place of SEQ ID NO.1 as CDR-L3.

Description

  The present invention relates to an anti-morphine antibody, a method for measuring morphine, and a morphine measurement kit including an anti-morphine antibody. More specifically, the present invention relates to an antibody that has a low ability to recognize heroin, cocaine, codeine, and ketamine and specifically recognizes morphine, a method for measuring morphine using the same, and a morphine measurement kit including the same.

  Unauthorized drug use that uses prescription drugs used for medical purposes in dosages other than medical purposes, or illegal use of drugs that are not medicines has been made for the purpose of obtaining a sense of euphoria and uplift. It is becoming a problem.

Such fraudulent drugs include amphetamine, methamphetamine, cocaine, ketamine, morphine, codeine, LSD, cannabis and the like. These drugs have psychotropic effects, cause hallucinations, delusions, and addiction.

Morphine is also a treatment drug, and there is a method of administration as one of the palliative treatments for patients with terminal cancer. In this method, it is necessary to balance the effects and side effects by adjusting the dose and blood concentration.

As an element for recognizing morphine, a polyclonal antibody having immunological reactivity has been prepared (Non-patent Document 1). In addition, immunochemical measurement methods (non-patent document 2 and patent document 1) using monoclonal antibodies having immunological reactivity to morphine are known, but those having high specificity for morphine are stable. It was difficult to get to.

Japanese Patent Laid-Open No. 2-86794

European Journal of Pharmacology, 38, 149-156, 1976 Molecular Immunology, 25, 937-943, 1988

  An object of the present invention is to provide an anti-morphine antibody that specifically recognizes morphine and has low reactivity with substances such as codeine, cocaine, ketamine, and heroin.

A further object of the present invention is to provide a method for measuring morphine in a sample using such an anti-morphine antibody and a kit for measuring morphine.

As a result of intensive investigations aimed at achieving the above object, the present inventors have succeeded in producing a novel anti-morphine antibody fragment and completed the present invention.
That is, the present invention relates to an anti-morphine antibody molecule comprising a heavy chain variable region and a light chain variable region that specifically recognizes morphine and has a low ability to recognize heroin, codeine, cocaine, and ketamine, and comprises a Fab fragment. , Fab ′ fragment, F (ab ′) ₂ fragment, single chain antibody (scFv), dimerization variable region (V region) fragment (Diabody), disulfide stabilized V region fragment (dsFv) and complementarity determining region Selected from the group consisting of an antibody fragment selected from peptides containing (CDR), and a complete antibody, and in the heavy chain variable region, a) described as positions 31 to 35 of SEQ ID NO: 1 in the sequence listing as CDR-H1 B) an amino acid sequence described in positions 50 to 65 of SEQ ID NO: 1 as CDR-H2, and c) an amino acid sequence described in positions 98 to 110 of SEQ ID NO: 1 as CDR-H3, Morphine antibody.

The present invention also relates to an anti-morphine antibody molecule comprising a heavy chain variable region and a light chain variable region that specifically recognizes morphine and has a low ability to recognize heroin, codeine, cocaine, ketamine, and a Fab fragment. , Fab ′ fragment, F (ab ′) ₂ fragment, single chain antibody (scFv), dimerization variable region (V region) fragment, disulfide stabilized V region fragment (dsFv) and complementarity determining region (CDR) In the light chain variable region, a) an amino acid sequence described in positions 160 to 174 of SEQ ID NO: 1 in the sequence listing as selected from the group consisting of an antibody fragment selected from a peptide comprising B) an anti-morphine antibody having the amino acid sequence of positions 190 to 196 of SEQ ID NO: 1 as CDR-L2, and c) the amino acid sequence of positions 229 to 237 of SEQ ID NO: 1 as CDR-L3, About.

The anti-morphine antibody is an anti-morphine antibody molecule comprising a heavy chain variable region and a light chain variable region that specifically recognizes morphine and has a low ability to recognize heroin, codeine, cocaine, and ketamine, Fragment, Fab ′ fragment, F (ab ′) ₂ fragment, single chain antibody (scFv), dimerization variable region (V region) fragment, disulfide stabilized V region fragment (dsFv) and complementarity determining region (CDR) ) Selected from the group consisting of an antibody fragment selected from a peptide containing a complete antibody, and a complete antibody, and in the heavy chain variable region, a) an amino acid described at positions 31 to 35 of SEQ ID NO: 1 in the sequence listing as CDR-H1 B) an amino acid sequence described in positions 50 to 65 of SEQ ID NO: 1 as CDR-H2, and c) an amino acid sequence described in positions 98 to 110 of SEQ ID NO: 1 as CDR-H3. In the chain variable region, a) sequence as CDR-L1 The amino acid sequence from position 160 to position 174 of SEQ ID NO: 1, b) the amino acid sequence from position 190 to position 196 of SEQ ID NO: 1 as CDR-L2, and c) the position 229 of SEQ ID NO: 1 as CDR-L3 To the amino acid sequence described at position 237.

The heavy chain variable region includes a polypeptide represented by positions 1 to 121 of SEQ ID NO: 1 in the sequence listing and a polypeptide in which 1 to several amino acids in the sequence are substituted, deleted, added, inserted, The light chain variable region is a polypeptide selected from positions 137 to 248 of SEQ ID NO: 1 in the sequence listing, and 1 to several amino acids in this sequence are substituted. It is preferably a polypeptide selected from the group consisting of a polypeptide having a deletion, addition or insertion.

  The anti-morphine antibody can be a single chain antibody comprising a heavy chain variable region-linker sequence-light chain variable region.

  The anti-morphine single chain antibody is a polypeptide consisting of the polypeptide represented by SEQ ID NO: 1 in the sequence listing and a polypeptide in which one to several amino acids in the sequence are substituted, deleted, added or inserted. It may contain a selected polypeptide.

  The present invention also holds a bacteriophage capable of presenting the above-mentioned anti-morphine single chain antibody, and has been deposited at the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center under the receipt number FERM ABP-11378. About E. coli.

The present invention also relates to an anti-morphine single chain antibody produced by the bacteriophage retained by the above-mentioned E. coli.

The present invention also relates to an antibody-like molecule that specifically recognizes morphine and has a low ability to recognize heroin, codeine, cocaine, and ketamine, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable In the region, a) an amino acid sequence described in positions 31 to 35 of SEQ ID NO: 1 in the sequence listing as CDR-H1, b) an amino acid sequence described in positions 50 to 65 of SEQ ID NO: 1 as CDR-H2, and c A) has the amino acid sequence from positions 98 to 110 of SEQ ID NO: 1 as CDR-H3, and in the light chain variable region, a) is described from positions 160 to 174 of SEQ ID NO: 1 in the sequence listing as CDR-L1 B) an amino acid sequence described in positions 190 to 196 of SEQ ID NO: 1 as CDR-L2, and c) an amino acid sequence described in positions 229 to 237 of SEQ ID NO: 1 as CDR-L3, In addition, another functionality Linked to the peptide, antibody-like molecule, on.

In the antibody-like molecule, the heavy chain variable region is a polypeptide represented by positions 1 to 121 of SEQ ID NO: 1 in the sequence listing and 1 to several amino acids in this sequence are substituted, deleted, added, inserted. The light chain variable region is a polypeptide represented by positions 137 to 248 of SEQ ID NO: 1 in the sequence listing and 1 to several of the sequences. The polypeptide may be a polypeptide selected from the group consisting of substitutions, deletions, additions, and inserted polypeptides.

  The present invention also provides an anti-morphine single chain antibody comprising a heavy chain variable region-linker sequence-light chain variable region that specifically recognizes morphine and has a low ability to recognize heroin, codeine, cocaine, ketamine, About.

  The present invention also relates to a morphine measurement kit comprising any of the above anti-morphine antibodies or antibody-like molecules.

The present invention also relates to a method for measuring morphine, which comprises the step of bringing any of the above anti-morphine antibodies or antibody-like molecules into contact with a sample.

The present invention is also a method for preparing the anti-morphine antibody described above,
(A) immunizing an animal with a complex of morphine and a specific protein;
(B) From the spleen cells of the immunized animal, using a complex of a protein different from the specific protein and morphine, the target cells are selected by binding to the complex, and a phage antibody library is prepared. And (c) using a complex of a protein different from the specific protein in step (a) and morphine, and selecting the phage having the antibody of interest from the phage antibody library by binding to the complex And a process for preparing the same. Here, the proteins used in steps (b) and (c) may be of the same type or different types.

  By using the anti-morphine antibody or anti-morphine antibody-like molecule of the present invention, only morphine can be specifically detected, and morphine can be measured quickly and accurately.

FIG. 4 is a graph showing binding properties to morphine and binding properties to ketamine, cocaine, heroin, codeine, and thyroblobulin for an anti-morphine single chain antibody of an example of the present invention. It is a figure which shows reacting with morphine-Tg according to the density | concentration of a morphine single chain antibody about the anti- morphine single chain antibody of an example of this invention. FIG. 3 is a graph showing that the concentration-dependent binding of a morphine single chain antibody is shown only to morphine for an example anti-morphine single chain antibody of the present invention. It is a figure showing that the binding dependent on a morphine concentration is shown about the anti-morphine single chain antibody of an example of this invention. FIG. 3 is a diagram comparing the binding properties of various anti-morphine single chain antibodies of the present invention to various substances with those of other companies' products or the hybridoma method.

The anti-morphine antibody referred to in the present invention is an antibody that specifically recognizes morphine and has a low ability to recognize heroin, cocaine, codeine, and ketamine, and is Fab, Fab ′, F (ab ′) ₂ , single antibody A molecule comprising an antibody fragment selected from a peptide comprising a chain antibody (scFv), a dimerization variable region (V region) fragment (Diabody), a disulfide stabilized V region fragment (dsFv) and a complementarity determining region (CDR); And all forms of the complete antibody molecule.

Here, morphine is (5α, 6α) -7,8-didehydro-4,5-epoxy-17-methylmorphinan-3,6-diol (CAS number 57-27-2), and is represented by the following chemical formula. It is.

Heroin is (5α, 6α) -7,8-didehydro-4,5-epoxy-17-methylmorphinan-3,6-diol diacetate, cocaine is (1R, 2R, 3S, 5S) -3- (Benzoyloxy) -8-methyl-8-azabicyclo [3.2.1] methyl octane-2-carboxylate, codeine is (5R, 6S) -7,8-didehydro-4,5-epoxy-3-methoxy-N -Methylmorphinan-6-ol, ketamine is (RS) -2- (2-chlorophenyl) -2-methylamino-cyclohexane-1-one.

Here, “specifically recognize morphine” means that the binding ability to morphine is significantly higher than the binding ability to other compounds, and preferably the Kd to morphine is 10 ⁻⁸ or less. It has a certain property. “Low recognition ability” means that the difference from the binding ability to other compounds is within 2 times, and preferably the Kd for the compound is 10 ⁻⁵ or more.

Furthermore, antibody-like molecules obtained by linking heavy chain variable regions and light chain variable regions to other functional polypeptides are also used in the present invention as equivalents of anti-morphine antibodies.

Here, a complete molecular monoclonal antibody refers to an antibody comprising a total of four polypeptide chains, two identical H chains and two identical L chains. Normally, the heavy chain and light chain are Y-shaped. They are arranged and linked by disulfide bonds (SS bonds). It can be obtained by fusing a heavy chain variable region and a light chain variable region with an immunoglobulin constant region.

In the present specification, the heavy chain variable region and the light chain variable region refer to a variable region (VH / VL) that is low in amino acid sequence similarity compared to other molecules, that is, specifically to morphine. This refers to the binding site.

In this specification, the heavy chain variable region-linker sequence-light chain variable region refers to a state in which such a heavy chain variable region and a light chain variable region are linked by a linker sequence, and the linker is not particularly limited. It may be any sequence of about 10 to 25 amino acid residues.

  The heavy chain variable region is: a) an amino acid sequence from position 31 to position 35 in SEQ ID NO: 1 in the sequence listing as CDR-H1, and b) an amino acid from position 50 to position 65 in SEQ ID NO: 1 as CDR-H2. And c) CDR-H3 preferably has the amino acid sequence described at positions 98 to 110 of SEQ ID NO: 1.

  The light chain variable region is a) an amino acid sequence from positions 160 to 174 in SEQ ID NO: 1 in the sequence listing as CDR-L1, and b) an amino acid from positions 190 to 196 in SEQ ID NO: 1 as CDR-L2. Preferably c) CDR-L3 having the amino acid sequence described from position 229 to position 237 of SEQ ID NO: 1.

More preferably, in the heavy chain variable region, the polypeptide represented by positions 1 to 121 of SEQ ID NO: 1 in the sequence listing and 1 to several amino acids in this sequence are substituted, deleted, added, or inserted. A polypeptide selected from the group consisting of:
The light chain variable region includes a polypeptide represented by positions 137 to 248 of SEQ ID NO: 1 in the sequence listing, and a polypeptide in which one to several amino acids in the sequence are substituted, deleted, added, inserted, A polypeptide selected from the group consisting of:

In the heavy chain variable region-linker sequence-light chain variable region of the present invention, the polypeptide represented by SEQ ID NO: 1 in the sequence listing and one to several amino acids in this sequence are substituted, deleted, added, or inserted. Particularly preferred is a polypeptide selected from the group consisting of, but not limited to.
Although the heavy chain variable region-linker sequence-light chain variable region of the present invention is not limited, it is particularly preferably a polypeptide encoded by the nucleic acid sequence represented by SEQ ID NO: 2 in the Sequence Listing.

In the present invention, when an antibody-like molecule is provided, the functional polypeptide to be linked is not limited. For example, g3p, CFP (cyan fluorescent protein), YFP (yellow fluorescent protein), GFP, BFP (blue fluorescent proteins), fluorescent dyes such as Qdot, His-Tag, E-Tag, HA-Tag, myc-Tag, FLAG-Tag and other tags, alkaline phosphatase (ALP), peroxidase (HRP) and other enzymes Examples thereof include proteins.

The anti-morphine antibody of the present invention can be preferably produced by a phage display method. Phage display methods are widely used to create large libraries of antibody fragments, taking advantage of the ability of bacteriophages to express and display biologically functional protein molecules on their surface. Various bacteriophage antibody display libraries and lambda phage expression libraries have been proposed.

In the present invention, in addition to an antibody phage library constructed on the basis of antibody genes derived from spleen cells of animals previously immunized with an antigen containing morphine, any library such as an intact naive library or a synthetic library It is also possible to use. In the case of an antibody phage library constructed on the basis of antibody genes derived from spleen cells of animals immunized with antigens containing morphine, the cells should be selected in a complex with morphine using the same protein as the immune antigen. However, it is also preferable to use a complex with morphine using a protein different from the immunizing antigen.

  In constructing the library, it is also preferable to design specific primers and amplify the VH region and the VL region separately. At this time, although not limited, for example, 19 VH fragment forward primers (SEQ ID NOs: 3 to 21 in the sequence listing), 4 reverse primers (SEQ ID NOs: 22 to 25 in the sequence listing), and VL fragment forward primer 18 (SEQ ID NO: 26 to 43 in the sequence listing) and 4 reverse primers (SEQ ID NO: 44 to 47 in the sequence listing) are preferably used. Antibody genes are generated by inserting into M13 or fd phage vectors or phagemid vectors. The target gene is expressed, for example, at the N-terminus of the gene 3 product (a kind of phage coat protein).

As a result, a peptide library containing peptides corresponding to the variable region of the desired anti-morphine antibody can be constructed. The phage library is then screened using the immobilized antigen and the specifically bound phage particles are recovered and amplified by infection into E. coli host cells. The antigen used at this time is preferably a complex with morphine using a protein different from the immune antigen. Anti-morphine antibody molecules of interest are enriched for reactive phage particles by immobilized antigen and / or labeled for plaque or colony lift screening.

In the present invention, it is particularly preferable to retain a bacteriophage capable of presenting an anti-morphine single chain antibody and deposit it with the receipt number FERM ABP-11378 at the National Institute of Advanced Industrial Science and Technology (AIST). E. coli is exemplified.

The heavy chain variable region and the light chain variable region of the present invention can be combined with a human-derived constant region to produce a chimeric molecule.

  Furthermore, the complementarity-determining region (CDR) of the present invention, that is, a) the amino acid sequence described from position 31 to position 35 of SEQ ID NO: 1 in the sequence listing as CDR-H1, and b) the sequence as CDR-H2 The amino acid sequence described from position 50 to position 65 of No. 1 and c) the amino acid sequence described from position 98 to position 110 of SEQ ID NO: 1 as CDR-H3, and / or a) the sequence number of the sequence listing as CDR-L1 1) the amino acid sequence from positions 160 to 174, b) the amino acid sequence from positions 190 to 196 of SEQ ID NO: 1 as CDR-L2, and c) the positions 229 to 237 of SEQ ID NO: 1 as CDR-L3 It is also possible to produce a humanized antibody by fusing the amino acid sequence described in 1 with a human-derived framework region.

  In preparing such a chimeric molecule or humanized antibody, a recombinant DNA encoding a fragment of the chimeric molecule, for example, a recombinant DNA encoding an Fab, Fab 'or Fv fragment of an anti-morphine antibody, conjugation of the antibody or fragment A recombinant DNA encoding a protein comprising the antibody, light chain variable region or heavy chain variable region of the antibody, and expressed in an appropriate expression system.

  The anti-morphine antibody or anti-morphine antibody-like molecule of the present invention can be preferably used for qualitative or quantitative measurement of morphine in a sample. Here, the sample may be blood, saliva, tissue, cerebrospinal fluid, tears, semen, urine, stool, pus, skin or mucosal secretions.

  In the present invention, morphine can be easily detected using the anti-morphine antibody or anti-morphine antibody-like molecule thus obtained, and can be suitably used for a morphine measurement method. Further, a kit containing an anti-morphine antibody or an anti-morphine antibody-like molecule, depending on the measurement method, a labeled secondary antibody or a labeled morphine hapten compound, a buffer, a detection reagent and / or a standard solution of morphine, etc. Can also be included. Preferred kits can be used for ELISA and other measurement methods using a label. When direct competitive inhibition ELISA is used, immobilized anti-morphine antibody or anti-morphine antibody-like molecule and antibody are retained. Carriers, enzymes, colloidal gold, antigens labeled by other means, detection reagents, and the like.

The method for measuring morphine is not particularly limited as long as it uses a normal antigen-antibody reaction. Radioisotope immunoassay (RIA), enzyme immunoassay (EIA), fluorescence or luminescence detection method, aggregation Method, immunoblot method, immunochromatography method and the like (Meth. Enzymol., 92, 147-523 (1983), Antibodies Vol. II IRL Press Oxford (1989)). Examples of labeling means include enzymes, gold colloids, radioisotopes, fluorescent materials, and luminescent materials. The radioisotope is not particularly limited, but for example, [ ¹²⁵ I], [ ¹³¹ I], [ ³ H], [ ¹⁴ C] and the like are preferable. Although it does not specifically limit as an enzyme, A stable and large specific activity is preferable, for example, (beta) -galactosidase, (beta) -glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase etc. are mentioned. Although it does not specifically limit as a fluorescent substance, For example, a fluorescamine, a fluorescein isothiocyanate, etc. are mentioned. Although it does not specifically limit as a luminescent substance, For example, luminol, a luminol derivative, luciferin, lucigenin etc. are mentioned. Among these, from the viewpoints of sensitivity and simplicity, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, ELISA using malate dehydrogenase, or immunochromatography using colloidal gold is preferable.

  Representative detection methods using ELISA include indirect competitive inhibition ELISA and direct competitive inhibition ELISA. For example, morphine can be detected by direct competitive inhibition ELISA using the anti-morphine antibody or anti-morphine antibody-like molecule of the present invention as described below.

(1) The anti-morphine antibody or anti-morphine antibody-like molecule of the present invention is immobilized on a carrier. The carrier to be used is preferably a microtiter plate having 96 holes, 48 holes, 192 holes or the like. For immobilization, for example, a buffer containing the immobilization antibody may be placed on a carrier and incubated.

(2) In order to prevent non-specific adsorption of the protein to the solid phase surface of the carrier, the solid phase surface portion where the antibody for immobilization is not adsorbed is blocked with a protein unrelated to the antibody. As a blocking agent, BSA or a skim milk solution can be used.

(3) A mixture is prepared by adding an enzyme-bound hapten obtained by binding a morphine hapten compound and an enzyme to a sample containing morphine at various concentrations. The enzyme-linked hapten may be prepared by any method without particular limitation as long as it is a method of binding a morphine hapten compound to an enzyme.

  The mixture of step (3) is reacted with the antibody-immobilized carrier obtained in step (2). A complex between these and a solid-phase support is generated by a competitive inhibition reaction between morphine and an enzyme-linked hapten. Since morphine is water-soluble, an aqueous solution can be used as a reaction solution. After completion of the reaction, the carrier is washed with a buffer to remove the enzyme-bound hapten that did not bind to the immobilized antibody. By detecting the amount of the immobilized antibody-enzyme-bound hapten complex, the amount of morphine in the sample is determined from a calibration curve prepared in advance.

(5) The amount of morphine can be calculated from the calibration curve by adding a chromogenic substrate solution that reacts with the labeling enzyme bound to the carrier and detecting the absorbance. When peroxidase is used as the labeling enzyme, for example, a chromogenic substrate solution containing hydrogen peroxide and 3,3 ′, 5,5′-tetramethylbenzidine or o-phenylenediamine can be used. Usually, after adding a chromogenic substrate solution and reacting at room temperature for about 10 minutes, the enzyme reaction is stopped by adding sulfuric acid. When 3,3 ', 5,5'-tetramethylbenzidine is used, absorbance at 450 nm is detected. When o-phenylenediamine is used, the absorbance at 492 nm is detected. In order to correct the background value, it is desirable to detect the absorbance at 630 nm at the same time.

  When alkaline phosphatase is used as the labeling enzyme, for example, a method of developing color using p-nitrophenyl phosphate as a substrate, adding an NaOH solution to stop the enzyme reaction, and detecting the absorbance at 415 nm can be mentioned.

  For the absorbance of the reaction solution to which morphine is not added, the decrease rate of the absorbance of the solution reacted with the antibody by adding morphine is calculated as the inhibition rate. The concentration of morphine in the sample can be calculated using a calibration curve prepared in advance based on the inhibition rate of the reaction solution to which a known concentration of morphine is added.

In another embodiment, morphine can be detected by indirect competitive inhibition ELISA.
In the detection method of the present invention, the sample can be pretreated according to the detection target and used as a sample, and then subjected to a direct competitive inhibition ELISA step or an indirect competitive inhibition ELISA. For most samples, any method that can extract morphine can be used. A sample containing morphine can be prepared as it is or after dilution with a buffer.

  In addition to the anti-morphine antibody or anti-morphine antibody-like molecule of the present invention, the kit for measuring morphine of the present invention can be suitably used in such a measurement method, if desired, a labeling enzyme, a secondary antibody, a buffer, Includes instructions.

  Furthermore, morphine can also be detected using an affinity column carrying the anti-morphine antibody or anti-morphine antibody-like molecule of the present invention.

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples. In each example, “%” is based on weight except for 0.1% Tween 20 and 10% diethanolamine.

Example 1
BSA complex and thyroglobulin (Tg) complex of drugs (morphine, heroin, codeine, cocaine, ketamine) were prepared using the following three methods.

Morphine was synthesized by adding formaldehyde to a mixture of morphine and activated BSA using Imject PharmaLink Immunogen Kit (Thermo Fisher Scientific). Thereafter, impurities were removed by a spin column to obtain a hapten-protein complex.

Separately, for morphine, formaldehyde was added to a mixture of morphine and amine to introduce an aminomethyl group (CH ₂ —NRH), which was reacted with bromoacetic acid benzyl ester. The catalyst was removed by filtration, and the solvent was distilled off under reduced pressure to obtain the desired carboxylic acid form (hapten-COOH). The carboxylic acid compound was dissolved in a small amount of DMF, NHS and EDC were added, and the mixture was stirred for 3 hours. This was added to the protein solution dissolved in the phosphate buffer and stirred overnight. Impurities were removed from the mixed solution by a spin column to obtain a hapten-protein complex.

For heroin, formaldehyde was added to a mixture of morphine and amine to introduce an aminomethyl group (CH ₂ —NRH), which was reacted with bromoacetic acid benzyl ester. An acetyl group was introduced into the OH group with acetic anhydride to convert it into a heroin derivative, and then the benzyl group was cleaved by catalytic hydrogen reduction. The catalyst was removed by filtration, and the solvent was distilled off under reduced pressure to obtain the desired carboxylic acid form (hapten-COOH). The carboxylic acid compound was dissolved in a small amount of DMF, NHS and EDC were added, and the mixture was stirred for 3 hours. This was added to the protein solution dissolved in the phosphate buffer and stirred overnight. Impurities were removed from the mixed solution by a spin column to obtain a hapten-protein complex.

For codeine, formaldehyde was added to a mixture of morphine and amine to introduce an aminomethyl group (CH ₂ —NRH), which was reacted with bromoacetic acid benzyl ester. A methyl group was introduced into the OH group with a methylating reagent to convert it to a codeine derivative, and then the benzyl group was cleaved by catalytic hydrogen reduction. The catalyst was removed by filtration, and the solvent was distilled off under reduced pressure to obtain the desired carboxylic acid form (hapten-COOH). The carboxylic acid compound was dissolved in a small amount of DMF, NHS and EDC were added, and the mixture was stirred for 3 hours. This was added to the protein solution dissolved in the phosphate buffer and stirred overnight. Impurities were removed from the mixed solution by a spin column to obtain a hapten-protein complex.

For morphine, N-CH ₃ was converted to NH (de-N-methylation) by heating the morphine in methanol after the 1-chloroethyl chloroformate reaction. Next, after reacting bromoacetic acid benzyl ester, the benzyl group was cleaved by catalytic hydrogen reduction. The catalyst was removed by filtration, and the solvent was distilled off under reduced pressure to obtain the desired carboxylic acid form (hapten-COOH). The carboxylic acid compound was dissolved in a small amount of DMF, NHS and EDC were added, and the mixture was stirred for 3 hours. This was added to the protein solution dissolved in the phosphate buffer and stirred overnight. Impurities were removed from the mixed solution by a spin column to obtain a hapten-protein complex.

Regarding heroin, N-CH ₃ was converted to NH (de-N-methylation) by heating morphine in 1-chloroethyl chloroformate followed by heating in methanol. Next, after reacting bromoacetic acid benzyl ester, an acetyl group was introduced into OH group with acetic anhydride to convert it into a heroin derivative, and the benzyl group was cleaved by catalytic hydrogen reduction. The catalyst was removed by filtration, and the solvent was distilled off under reduced pressure to obtain the desired carboxylic acid form (hapten-COOH). The carboxylic acid compound was dissolved in a small amount of DMF, NHS and EDC were added, and the mixture was stirred for 3 hours. This was added to the protein solution dissolved in the phosphate buffer and stirred overnight. Impurities were removed from the mixed solution by a spin column to obtain a hapten-protein complex.

After the reaction of codeine with 1-chloroethyl chloroformate, N-CH ₃ was converted to NH by heating in methanol (de-N-methylation), then reacted with bromoacetic acid benzyl ester, and the benzyl group was cleaved by catalytic hydrogen reduction. The catalyst was removed by filtration, and the solvent was distilled off under reduced pressure to obtain the desired carboxylic acid form (hapten-COOH). The carboxylic acid compound was dissolved in a small amount of DMF, NHS and EDC were added, and the mixture was stirred for 3 hours. This was added to the protein solution dissolved in the phosphate buffer and stirred overnight. Impurities were removed from the mixed solution by a spin column to obtain a hapten-protein complex.

Ketamine was dissolved in 1,2-dichloroethane together with bromoacetic acid benzyl ester and heated to reflux in the presence of sodium carbonate overnight. Water was added to the reaction solution to separate the organic layer, washed with water and dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The crystalline residue was dissolved in ethyl alcohol, palladium carbon was used as a catalyst, and the mixture was stirred overnight at room temperature in a hydrogen atmosphere to reductively cleave the benzyl group. The catalyst was removed by filtration, and the solvent was distilled off under reduced pressure to obtain the desired carboxylic acid form (hapten-COOH). The carboxylic acid compound was dissolved in a small amount of DMF (N, N-dimethylformamide), NHS (N-hydroxysuccinimide) and EDC (water-soluble carbodiimide) were added, and the mixture was stirred for 3 hours. This was added to the protein solution dissolved in the phosphate buffer and stirred overnight. Impurities were removed from the mixed solution by a spin column to obtain a hapten-protein complex.

By reacting cocaine with 1-chloroethyl chloroformate and heating in methanol, N-CH ₃ was converted to NH (de-N-methylation). Thereafter, it was dissolved in 1,2-dichloroethane together with bromoacetic acid benzyl ester, and heated under reflux overnight in the presence of sodium carbonate. Water was added to the reaction solution to separate the organic layer, washed with water and dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The crystalline residue was dissolved in ethyl alcohol, palladium carbon was used as a catalyst, and the mixture was stirred overnight at room temperature in a hydrogen atmosphere to reductively cleave the benzyl group. The catalyst was removed by filtration, and the solvent was distilled off under reduced pressure to obtain the desired carboxylic acid form (hapten-COOH). The carboxylic acid compound was dissolved in a small amount of DMF, NHS and EDC were added, and the mixture was stirred for 3 hours. This was added to the protein solution dissolved in the phosphate buffer and stirred overnight. Impurities were removed from the mixed solution by a spin column to obtain a hapten-protein complex.

(Example 2)
All hapten-protein complexes obtained in Example 1 were separately immunized at BALB / cCrlCrlj, female, 7 weeks old. For the first immunization, a mixture of Adjuvant, Complete (Freund) (DIFCO) and a hapten-protein complex is administered into the peritoneal cavity. For the second and subsequent immunizations, Adjuvant, Incomplete (Freund) (DIFCO) and a hapten-protein complex are administered. The mixture with the body was administered intraperitoneally. Immunization was carried out until the antibody titer sufficiently increased at intervals of about 2 to 3 weeks. The antibody titer was measured by ELISA.

A hapten complexed with a protein that was not used as an immunogen was applied to an ELISA plate (1 ng / μl) and 50 μl was reacted overnight. After washing, blocking was performed with 200 μl of 1% Block Ace (DS Pharma Biomedical Co., Ltd.) for 2 hours. After washing, 50 μl of serially diluted serum sample was reacted for 2 hours. After washing, 50 μl of HRP-anti mouse IgG (Santa Cruz) was reacted for 30 minutes. After washing, 100 μl of o-phenylenediamine dihydrochloride (Sigma FAST) 50set (sigma) was reacted for 30 minutes, the reaction was stopped with 50 μl of 2M (10%) H ₂ SO ₄ and measured at 490 nm.

(Example 3)
Next, a mouse scFv library was constructed (scFv: single chain variable fragment).

Spleen cells were collected from the spleen of BALB / cCrlCrlj immunized with a complex of morphine and protein, reacted with dish that had immobilized 20 μg of morphine-Tg for 30 minutes, and cells that bind to morphine-Tg were selected. EPICS XL (BECKMAN COULTER) was used for analysis.

Here, the complex of morphine and protein used for immunization was prepared as a mixture of morphine and activated BSA using Imject PharmaLink Immunogen Kit (Thermo Fisher Scientific) among the complexes obtained in Example 1. It was synthesized by adding formaldehyde and then removed by a spin column to remove impurities.

The morphine-Tg used for immobilization is the morphine-Tg complex obtained in Example 1. That is, first, formaldehyde was added to a mixture of morphine and amine to introduce an aminomethyl group (CH ₂ —NRH), and this was reacted with bromoacetic acid benzyl ester. The catalyst was removed by filtration, and the solvent was distilled off under reduced pressure to obtain the desired carboxylic acid form (hapten-COOH). The carboxylic acid compound was dissolved in a small amount of DMF, NHS and EDC were added, and the mixture was stirred for 3 hours. This was added to the protein solution dissolved in the phosphate buffer and stirred overnight. Impurities were removed from the mixed solution by a spin column to obtain a hapten-protein complex.

  RNA was extracted from the cells that bound to the selected morphine-Tg to prepare cDNA. RNA was extracted from the cells binding to the selected morphine-Tg by ISOGEN (Nippon Gene), and cDNA was prepared by First-Strand cDNA Synthesis Kit (GE Healthcare). From this, VH fragments and VL fragments were prepared using mouse antibody gene-specific primers. Based on the primer design published by JOrg Burmester, etc., primers were used which were modified in several places including restriction enzyme sites. 19 forward primers for the VH fragment (SEQ ID NO: 3 to 21 in the sequence listing), 4 reverse primers (SEQ ID NO: 22 to 25 in the sequence listing), 18 forward primers for the VL fragment (SEQ ID NO: 26 in the sequence listing) ˜43) and 4 reverse primers (SEQ ID NOs: 44 to 47 in the sequence listing).

Next, VH fragments and VL fragments were prepared by cutting out the target fragment from an electrophoresis gel and purifying it with DEAE paper. In addition, a part of the linker is incorporated into the reverse primer for VH fragment production and the forward primer for VL fragment production, and it is created so that it anneals at one central part of (Gly) 3Ser repeat sequences. Has been. The purified VH and VL fragments were attached with restriction enzyme sites sfiI and speI, 5′-c tat gcg gcc cag ccg gcc (positions 1 to 19 in SEQ ID NO: 3 in the sequence listing) as a forward primer, and 5 as a reverse primer. Using '-gtg gtg act agt gcc acc ac (positions 1 to 20 in SEQ ID NO: 44 in the sequence listing), the fragments were ligated by PCR to obtain scFv. After digesting the scFv and pCANTAB5E phagemid vector with 50-100 units of sfiI (NEB), 50 ° C, 8 hours, 50 units of speI (NEB), 37 ° C, overnight, excise the target fragment from the electrophoresis gel, The fragments were digested with restriction enzymes and purified with DEAE paper. The scFv and the pCANTAB5E phagemid vector were ligated overnight at 16 ° C. with 200 units of T4 DNA ligase (NEB) at a molar ratio of 3: 1. After purification of the ligation product, E. coli TG1 Electrocompetent Cells (Lucigen) were electroporated with BIO-RAD GENE PULSER ^R II under the conditions of 2.0 mm cuvette, 25 μF, 200Ω, 25 KV. A library was cultured overnight in 400 mL of 2TYAG (Bacto Tryptone 1.6%, Bacto Yeast Extract 1.0%, NaCl 0.5%, Glucose 2.0%, Ampicillin 0.01%). As a result, a 1.1X10 ⁸ diversity library was constructed.

Example 4
To rescue phage displaying scFv, approximately 10 ⁹ -10 ¹⁰ cfu glycerol library stock was infected with 10 ml of 2TYAG and cultured at 37 ° C. The cells were cultured until OD600 = 0.4-0.5 and infected with 10 ¹¹ cfu of M13 KO7 helper phage. After infection, the medium was replaced with 2TYAK (kanamycin 0.005%) medium and cultured overnight.

(Example 5)
Next, panning was performed. 1 μg of morphine-Tg / 0.1M NaHCO ₃ was immobilized in an immunotube by reacting at 4 ° C. overnight. Subsequently, after blocking with 1.0% Block Ace for 2 hours, the plate was washed 3 times with 0.1% Tween20-PBS (hereinafter abbreviated as PBST). To this, 0.55 mL (1.2 × 10 ¹² cfu / 1% Tg, 1% BSA solution) of a mouse-derived antibody phage library (single-chain variable region fragment (scFv) -displayed phage solution) was added and reacted.

Next, this reaction solution was washed 10 times with PBST and 5 times with PBS, and then 0.5 mL of glycine buffer (pH 2.2) was added to elute scFv-displayed phages that bind to morphine-Tg. 1M Tris (hydroxymethyl) aminomethane-HCl, (pH 9.0) was added to the eluted phage to adjust the pH, and then infected with E. coli TG1 in the logarithmic growth phase. A small amount of TG1 after infection was seeded on 2TYAG plates and cultured overnight at 30 ° C. (1st panning). The remaining TG1 was cultured overnight at 30 ° C. in 2TYAG to obtain a 1st panned library, and the above operations were repeated for 2nd panning and 3rd panning.
Panning was performed 3 times in total using the above-mentioned morphine-Tg immobilized plate. After the second and third panning, clones were arbitrarily extracted from the 2TYAG plate, and confirmed for scFv expression, specificity for morphine-Tg by ELISA (screening), and analysis of the base sequence. It was shown that scFv-displaying phages that react with morphine-Tg were enriched with the number of pannings.

(Example 6)
ELISA for screening isolated clones was performed by immobilizing morphine-Tg on an ELISA plate. Specifically, 50 ng / 50 μl of morphine-Tg or Tg was placed in an ELISA plate (Nunc) and allowed to stand overnight at room temperature for immobilization. The immobilization plate was blocked by placing 200 μL / well of a 1.0% block ace solution in an ELISA plate and allowing to stand at room temperature for 2 hours. Next, 50 μL / well of a sample solution containing scFv-displayed phage was added to the ELISA plate for reaction, and then the sample solution was discarded and washed 5 times with a washing solution. Subsequently, 50 μl of 1: 1000 diluted anti-M13 monoclonal antibody was added to the immobilized scFv phage and allowed to react for 1 hour, and 50 μl of 1: 1000 diluted alkaline phosphatase (AP) -labeled anti-mouse IgG antibody (CALTAG lab) was reacted as a secondary antibody for 30 minutes. After washing this reaction solution 5 times with a washing solution, add 50 μL / well of a chromogenic substrate solution (a solution containing 1 g / mL p-nitrophenyl phosphate (Wako) and 10% diethanolamine (Wako)), and shade it at room temperature. It was. As a result of measuring the absorbance at 405 nm with EnSpire 2300 Multilabel Reader (PerkinElmer), 6 clones specific to morphine-Tg were obtained.

(Example 7)
Escherichia coli HB1251 was infected with phages of scFv clones that react with morphine-Tg. SOBAG-N plate (Bacto Tryptone 2%, Bacto Yeast Extract 0.5%, NaCl 0.05%, KCl final concentration 2.5 mM, MgCl2 final concentration 10 mM, Glucose 2.0%, Ampicillin 0.01%, NaI final concentration 125 μg / ml, Agar 1.5%) These Escherichia coli cells were seeded at 30 ° C. overnight, and single colonies were pre-cultured in 2TYAG medium at 30 ° C. overnight. A part of this preculture was transplanted to 2TYAG and cultured at 37 ° C. until OD600 = 0.4-0.5. After centrifugation, this was replaced with 2TYAI medium (final concentration 1 mM IPTG, 0.01% ampicillin) and further cultured for 12 hours to induce scFv expression. After completion of the culture, centrifugation was performed to collect a culture supernatant fraction and a bacterial cell fraction. The cell fraction was suspended in PBS containing 1 mM EDTA, and the cells were left on ice for 30 minutes. Subsequently, the mixture was centrifuged at 15000 × rpm for 10 minutes, and the supernatant was collected, filtered through a 0.45 μm filter, and used as a starting material for purifying scFv from the periplasm fraction. Further, the precipitate was suspended in PBS, boiled for 5 minutes, centrifuged at 15000X rpm for 5 minutes, and the supernatant was used as the cytoplasm fraction. The expression level of each fraction was analyzed by dot blot. In the dot blot, the culture supernatant fraction, periplasm fraction, and cytoplasm fraction were blotted to Immobilon-P (Millipore Corporation), blocked with 1% Block Ace, 5 μg / ml anti-E-tag monoclonal antibody (ABNOVA), A 1: 2000 dilution of peroxidase (HRP) -labeled sheep polyclonal anti-mouse IgG (H + L) antibody was reacted as a secondary antibody. ECL (NACALAI TESQUE.INC) was used as a detection reagent, and detection was performed with LAS1000 (FUJIFILM). As a result, it was confirmed that scFv was highly expressed in the periplasm fraction in all the clones analyzed.

(Example 8)
Next, the DNA base sequences of the VH chain and VL chain gene of the isolated clone scFv gene were determined using BigDye Terminator v3.1 / 1.1 Cycle Sequencing kit (Applied Biosystems). As a result of ELISA and sequence analysis, all the isolated clones were identical. The gene sequence and amino acid sequence are shown (SEQ ID NO: 2 and 1 in the sequence listing).

Example 9
Purification of scFv The thus-prepared clone for purification was purified according to a conventional method using Ni-column (GE Healthcare). Elution was performed with 20 mM Na-phosphate, 500 mM NaCl, 500 mM imidazole, pH 7.4. After dialysis with PBS, confirmation was performed by SDS-PAGE. Of the periplasm fraction, the culture supernatant fraction, and the culture supernatant fraction, a sample of the fraction deposited during dialysis was used. SDS-PAGE was performed under 15% gel, 20 mM, 1 hour under non-reducing conditions. For staining, SimplyBlue SafeStain (Invitrogen) was used.

(Example 10)
Binding of purified scFv to morphine-Tg Next, the binding of purified scFv to morphine-Tg was measured by ELISA. 50 ng / 50 μl of morphine-Tg, ketamine-Tg, cocaine-Tg, heroin-Tg, codeine-Tg, Tg as a control on a 96-well plate (NUNC. MAXISORP) immobilized at room temperature with 1% block ace at room temperature After time blocking, 50 μL of purified scFv was added and reacted at room temperature for 2 hours. After washing 5 times with PBST, 50 μl of 1: 1000 diluted anti-E-tag polyclonal antibody was reacted at room temperature for 1 hour, and 50 μl of 1: 1000 diluted alkaline phosphatase (AP) -labeled sheep polyclonal anti-rabbit IgG as a secondary antibody. For 30 minutes at room temperature.
After washing 5 times with PBST, 50 μL / well of a chromogenic substrate solution (a solution containing 1 g / mL p-nitrophenyl phosphate (Wako) and 10% diethanolamine (Wako)) was added, and the color was allowed to develop at room temperature, protected from light. As a result of measuring the absorbance at 405 nm with the EnSpire 2300 Multilabel Reader (PerkinElmer), the periplasm fraction before and after dialysis (for EDTA removal), the purified sample, or the fraction purified from the culture supernatant, In any case, it had high reactivity to morphine-Tg. Moreover, it was low activity with respect to other ketamine, cocaine, heroin, codeine, and thyroglobulin (FIG. 1). In FIG. 1, pur / peri_5 μl represents a sample 5 μl purified from the periplasm fraction, sup / pur_5 μl represents a sample 5 μl purified from the culture supernatant fraction, (−) dialysis_50 μl represents a sample 50 μl before periplasm fraction dialysis (+) Dialysis_50 μl represents 50 μl of sample after periplasmic fraction dialysis, MMT is morphine-Tg, KT is ketamine-Tg, Cc-T is cocaine-Tg , Cd-T is codeine-Tg, and Tg is thyroglobulin. The numbers in the figure represent experimental sample serial numbers, and the antigen-M-Tg indicates that morphine is used as a starting material and a linker is bonded to a carbon atom of an aromatic ring.

Dynamic response to morphine-Tg of purified scFv
50 ng / 50 μl of morphine-Tg, 96-well plate (NUNC. MAXISORP) solidified with Tg as a control, blocked with 1% Block Ace for 2 hours at room temperature, and then added 50 μL of various concentrations of purified scFv at room temperature. The reaction was performed for 2 hours. After washing with PBST 5 times, in the case of scFv, 50 μl of 1: 1000 diluted anti-E-tag polyclonal antibody was reacted at room temperature for 1 hour, and 50 μl of 1: 1000 diluted peroxidase (HRP) -labeled sheep polyclonal was used as the secondary antibody. Anti-rabbit IgG was reacted at room temperature for 30 minutes. Washed 5 times with PBST, reacted with 100 μl of o-phenylenediamine dihydrochloride (Sigma FAST) 50set (sigma) for 30 minutes, stopped with 50 μl of 2M (10%) H 2 SO 4 and measured at 490 nm. As a result, it was shown that purified scFv reacts with morphine-Tg in a concentration-dependent manner (FIG. 2).

Evaluation of cross-reactivity of purified scFv
A 96-well plate (NUNC. MA) solidified with 50 ng / 50 μl of morphine-Tg, ketamine-Tg, cocaine-Tg, heroin-Tg, codeine-Tg, Tg as controls
XISORP) was blocked with 1% Block Ace for 2 hours at room temperature, 50 μL of various concentrations of purified scFv were added, and the mixture was reacted at room temperature for 2 hours. After washing 5 times with PBST, 50 μl of 1: 1000 diluted anti-E-tag polyclonal antibody was reacted at room temperature for 1 hour, and 50 μl of 1: 1000 diluted peroxidase (HRP) -labeled sheep polyclonal anti-rabbit IgG was used as the secondary antibody. The reaction was allowed to proceed for 30 minutes at room temperature. Washed 5 times with PBST, reacted with 100 μl of o-phenylenediamine dihydrochloride (Sigma FAST) 50set (sigma) for 30 minutes, stopped with 50 μl of 2M (10%) H ₂ SO ₄ and measured at 490 nm. As a result, the concentration-dependent binding of purified scFv was shown only to morphine-Tg. Almost no binding to the control drug (FIG. 3).

Drug concentration-dependent binding activity of purified scFv After blocking for 2 hours at room temperature with 1% Block Ace in 96-well plate (NUNC. MAXISORP) solidified with various concentrations of morphine-Tg, purification at 3 different constant concentrations 50 μL of scFv was added and reacted at room temperature for 2 hours. After washing 5 times with PBST, 50 μl of 1: 1000 diluted anti-E-tag polyclonal antibody was reacted at room temperature for 1 hour, and 50 μl of 1: 1000 diluted peroxidase (HRP) -labeled sheep polyclonal anti-rabbit IgG was used as the secondary antibody. The reaction was allowed to proceed for 30 minutes at room temperature. Washed 5 times with PBST, reacted with 100 μl of o-phenylenediamine dihydrochloride (Sigma FAST) 50set (sigma) for 30 minutes, stopped with 50 μl of 2M (10%) H 2 SO 4 and measured at 490 nm. As a result, the purified scFv showed a concentration-dependent binding property of morphine-Tg (FIG. 4).

Comparison with hybridomas and products from other companies
96-well plate (NUNC. MA) solidified with 50 ng / 50 μl of morphine-Tg, ketamine-Tg, cocaine-Tg, heroin-Tg, codeine-Tg, BSA, Tg as controls
XISORP) was blocked with 1% Block Ace at room temperature for 2 hours, and then purified scFv, an antibody prepared by the hybridoma method, and 50 μL of another company's product were added and reacted at room temperature for 2 hours. After washing 5 times with PBST, 50 μl of 1: 1000 diluted anti-E-tag polyclonal antibody was reacted at room temperature for 1 hour, and 50 μl of 1: 1000 diluted peroxidase (HRP) -labeled sheep polyclonal anti-rabbit IgG was used as the secondary antibody. The reaction was allowed to proceed for 30 minutes at room temperature. In the case of anti-Morphine Ab • product B, 50 μl of HRP-anti mouse IgG (Santa Cruz) was reacted at room temperature for 30 minutes. Washed 5 times with PBST, reacted with 100 μl of o-phenylenediamine dihydrochloride (Sigma FAST) 50set (sigma) for 30 minutes, stopped with 50 μl of 2M (10%) H 2 SO 4 and measured at 490 nm. As a result, it was shown that only scFv reacts specifically with morphine-Tg (FIG. 5). In FIG. 5, the abbreviations are the same as in FIG. New refers to an additionally synthesized antigen, and the performance is the same as that of a substance not labeled as new.

Calculation of kinetic parameters by SPR of purified scFv
Morphine-Tg was immobilized on a CM5 sensor chip (Amersham Biosciences). 62.5 ng / ml, 125 ng / ml, 250 ng / ml, 500 ng / ml and 1000 ng / ml of purified scFv were reacted and analyzed. As a result, the ka value was 3.72 × 10 ⁵ (1 / Ms), the kd value was 5.84 × 10 ⁻⁴ (1 / s), and the Kd value was 1.47 × 10 ⁻⁹ (M).

Structural analysis by molecular modeling
As a result of molecular modeling with PyMol, anti-morphine scFv was predicted to form cavities, which are structural features of antibodies against low molecular weight compounds, due to the long CDR H3 region. (CDR H1: cyan, CDR H2: magenta, CDR H3: yellow, CDR L1: blue, CDR L2: Red, CDR L3: orange)

  For the anti-morphine single chain antibody obtained in this example, germ-line usage was determined by database homology search by IMGT / V-QUEST. As a result, the heavy chain V gene was composed of IGHV5 (IMGT subgroup), and the light chain V gene was composed of IGKV3 (IMGT subgroup).

  The anti-morphine antibody of the present invention specifically recognizes morphine, has low reactivity to similar substances such as heroin, codeine, cocaine, and ketamine, and is easy to prepare derivatives for morphine measurement and morphine detection. Very useful.

Claims (14)

An anti-morphine antibody molecule comprising a heavy chain variable region and a light chain variable region that specifically recognizes morphine and has a low ability to recognize heroin, codeine, cocaine, and ketamine, comprising a Fab fragment, a Fab ′ fragment, Includes F (ab ') ₂ fragment, single chain antibody (scFv), dimerization variable region (V region) fragment (Diabody), disulfide stabilized V region fragment (dsFv) and complementarity determining region (CDR) Selected from the group consisting of an antibody fragment selected from peptides and a complete antibody,
In the heavy chain variable region, a) an amino acid sequence described from position 31 to position 35 in SEQ ID NO: 1 in the sequence listing as CDR-H1, and b) an amino acid described from position 50 to position 65 in SEQ ID NO: 1 as CDR-H2 An anti-morphine antibody having the sequence and c) the amino acid sequence of positions 98 to 110 of SEQ ID NO: 1 as CDR-H3. An anti-morphine antibody molecule comprising a heavy chain variable region and a light chain variable region that specifically recognizes morphine and has a low ability to recognize heroin, codeine, cocaine, and ketamine, comprising a Fab fragment, a Fab ′ fragment, Includes F (ab ') ₂ fragment, single chain antibody (scFv), dimerization variable region (V region) fragment (Diabody), disulfide stabilized V region fragment (dsFv) and complementarity determining region (CDR) Selected from the group consisting of an antibody fragment selected from peptides and a complete antibody,
In the light chain variable region, a) an amino acid sequence described in positions 160 to 174 of SEQ ID NO: 1 in the sequence listing as CDR-L1, and b) an amino acid described in positions 190 to 196 of SEQ ID NO: 1 as CDR-L2. An anti-morphine antibody having the sequence and c) the amino acid sequence of positions 229 to 237 of SEQ ID NO: 1 as CDR-L3. An anti-morphine antibody molecule comprising a heavy chain variable region and a light chain variable region that specifically recognizes morphine and has a low ability to recognize heroin, codeine, cocaine, and ketamine, comprising a Fab fragment, a Fab ′ fragment, Includes F (ab ') ₂ fragment, single chain antibody (scFv), dimerization variable region (V region) fragment (Diabody), disulfide stabilized V region fragment (dsFv) and complementarity determining region (CDR) Selected from the group consisting of an antibody fragment selected from peptides and a complete antibody,
In the heavy chain variable region, a) an amino acid sequence described from position 31 to position 35 in SEQ ID NO: 1 in the sequence listing as CDR-H1, and b) an amino acid described from position 50 to position 65 in SEQ ID NO: 1 as CDR-H2 The sequence, and c) the amino acid sequence described in positions 98 to 110 of SEQ ID NO: 1 as CDR-H3,
In the light chain variable region, a) an amino acid sequence described in positions 160 to 174 of SEQ ID NO: 1 in the sequence listing as CDR-L1, and b) an amino acid described in positions 190 to 196 of SEQ ID NO: 1 as CDR-L2. The anti-morphine antibody according to claim 1 or 2, which has the sequence, and c) the amino acid sequence of positions 229 to 237 of SEQ ID NO: 1 as CDR-L3. A polypeptide in which the heavy chain variable region is represented by positions 1 to 121 of SEQ ID NO: in the sequence listing and a polypeptide in which 1 to several amino acids in the sequence are substituted, deleted, added or inserted A polypeptide selected from the group consisting of:
A polypeptide in which the light chain variable region is represented by positions 137 to 248 of SEQ ID NO: 1 in the sequence listing, and a polypeptide in which one to several amino acids in the sequence are substituted, deleted, added, or inserted; A polypeptide selected from the group consisting of:
The anti-morphine antibody according to any one of claims 1 to 3.   The anti-morphine single chain antibody according to any one of claims 1 to 4, which is a single chain antibody comprising a heavy chain variable region-linker sequence-light chain variable region.   A polypeptide represented by SEQ ID NO: 1 in the sequence listing and a polypeptide selected from the group consisting of a polypeptide in which one to several amino acids in the sequence are substituted, deleted, added or inserted. 6. The anti-morphine single chain antibody according to 5.   E. coli having a bacteriophage capable of displaying the anti-morphine single-chain antibody according to claim 5 or 6, and having a receipt number of FERM ABP-11378. An anti-morphine single chain antibody produced by a bacteriophage carried by E. coli of claim 7. An antibody-like molecule that specifically recognizes morphine and has low ability to recognize heroin, codeine, cocaine, ketamine, comprising a heavy chain variable region and a light chain variable region, wherein a) As the CDR-H1, the amino acid sequence described in the 31st to 35th positions of SEQ ID NO: 1 in the sequence listing, b) The amino acid sequence described as the 50th to 65th position of SEQ ID NO: 1 as the CDR-H2, and c) As the CDR-H3 Having the amino acid sequence from position 98 to position 110 of SEQ ID NO: 1, and in the light chain variable region, a) the amino acid sequence of positions 160 to 174 of SEQ ID NO: 1 in the sequence listing as CDR-L1, b A) the amino acid sequence described in positions 190 to 196 of SEQ ID NO: 1 as CDR-L2, and c) the amino acid sequence described in positions 229 to 237 of SEQ ID NO: 1 as CDR-L3,
Furthermore, an antibody-like molecule linked to another functional peptide. A polypeptide in which the heavy chain variable region is represented by positions 1 to 121 of SEQ ID NO: 1 in the sequence listing, and a polypeptide in which one to several amino acids in the sequence are substituted, deleted, added or inserted; A polypeptide selected from the group consisting of:
A polypeptide in which the light chain variable region is represented by positions 137 to 248 of SEQ ID NO: 1 in the sequence listing, and a polypeptide in which one to several amino acids in the sequence are substituted, deleted, added, or inserted; A polypeptide selected from the group consisting of:
The antibody-like molecule according to claim 9.   An anti-morphine single chain antibody comprising a heavy chain variable region-linker sequence-light chain variable region that specifically recognizes morphine and has low ability to recognize heroin, codeine, cocaine, and ketamine.   A morphine measurement kit comprising the anti-morphine antibody or antibody-like molecule according to any one of claims 1 to 6 and 8 to 11. A method for measuring morphine, comprising a step of bringing the anti-morphine antibody or antibody-like molecule according to any one of claims 1 to 6 and 8 to 11 into contact with a sample. A method for preparing the anti-morphine antibody according to any one of claims 1 to 6,
(A) immunizing an animal with a complex of morphine and a specific protein;
(B) From the spleen cells of the immunized animal, using a complex of a protein different from the specific protein and morphine, the target cells are selected by binding to the complex, and a phage antibody library is prepared. And (c) using a complex of a protein different from the specific protein in step (a) and morphine, and selecting the phage having the antibody of interest from the phage antibody library by binding to the complex A preparation method comprising the steps of: